Recent discoveries have lead to the identification of many genetic, metabolic, and cellular processes essential for “normal” unaffected human development and the prevention of disease. These discoveries have charged the scientific community to begin to understand the molecular, cellular and physiological underpinnings of human disease. Combing modern technology with traditional approaches to basic scientific questions our department focuses on some of the most important areas of disease biology affecting the human condition today.

Michael CaterinaSolomon H. Snyder Professor of Neurosurgery
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

My lab studies mechanisms underlying pain sensation. One focus of the lab is a group of ion channel proteins of the Transient Receptor Potential Vanilloid (TRPV) family. These channels share the intriguing feature that they can be activated by warm or painfully hot temperatures, as well as by many nonthermal stimuli. For example, TRPV1, the founding member of this family, can be activated by painful heat (>42°C), by protons, or by pungent chemicals such as capsaicin. This channel is strongly expressed in nociceptive neurons and is essential for normal behavioral responses to noxious heat. By examining these channels in recombinant and native systems, and taking advantage of knockout mice lacking one or more subtypes, we are dissecting the biological contributions of these channels to pain sensation and other processes in both neuronal and nonneuronal cells. A second focus of the lab is the use of cutting-edge molecular, cellular, genetic, behavioral and physiological approaches to understand the biological and pathophysiological basis of chronic pain in animal models and in human disease.

Current areas of emphasis include:

Understanding the mechanisms by which keratinocytes in the skin contribute to "indirect" pain perception by signaling to epidermal nerve endings under healthy conditions and in painful disease states in both animal models and human patients.

Uncovering non-nociceptive roles for TRPV channels in epithelial cells of the skin and urinary bladder.

Developing novel assays and reagents for the quantification and characterization of acute nociception and chronic pain.

Dissecting the cellular and molecular events that contribute to neuropathic pain and exploring strategies to reverse this condition.

Ryuya FukunagaAssistant Professor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

Overview

The Fukunaga lab investigates the mechanism and biology of small silencing RNAs. We try to understand how small silencing RNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), are produced and how they function. We use a combination of biochemistry, biophysics, fly genetics, cell culture, X-ray crystallography and next-generation sequencing, in order to understand the biogenesis and function of small silencing RNAs from the atomic to the organismal level.

1. miRNA

miRNAs are 21-24 nt long RNA. In fruit fly Drosophila, miRNAs are transcribed as long primary transcripts called pri-miRNAs (Figure 1). The pri-miRNA is cleaved into pre-miRNA in the nucleus by the RNase III enzyme Drosha, aided by the dsRNA-binding partner protein Pasha. The Exportin-5/Ran-GTP complex transports pre-miRNA from the nucleus to the cytoplasm. In cytoplasm, Dicer-1, aided by the dsRNA-binding partner protein Loqs-PA or Loqs-PB, cleaves the pre-miRNA into miRNA duplex. miRNA is then loaded to Argonaute1 and binds target mRNAs through base complementarity of the miRNA sequence at positions 2-8 (called seed sequence). miRNA-Ago1 binding to the target mRNAs causes translational repression and mRNA degradation.

Loqs-PB, but not its alternative splicing isoform Loqs-PA, changes the nucleotide positions at which Dicer-1 cleaves pre-miRNA and produces miRNA with distinct length (Figure 2). These alternatively produced miRNAs can have distinct seed sequences and therefore regulate different target mRNAs. The mammalian Dicer partner protein TRBP, but not its paralogue PACT, changes the length and the seed sequence of miRNAs produced by Dicer in mammals. The Fukunaga lab investigates how Dicer partner proteins (Loqs-PB in fly and TRBP in mammals) change the miRNA length generated by the Dicer enzymes. We also try to uncover biological significance of the alternative miRNA production. Our hypothesis is that the alternative splicing of Loqs-PA/Loqs-PB in fly and the gene expression of TRBP/PACT in mammals are finely regulated in each tissue and developmental stage, leading to regulated production of distinct miRNA isoforms, and that such fine regulation is important for biology. For this end, we are trying to make miR-307a knockout flies and plan to analyze the molecular phenotypes. Furthermore, we are trying to discover novel factors and mechanisms regulating the miRNA production and function.

In another project, as collaboration with a physician scientist, Dr. Roselle Abraham at the Cardiology Division of Department of Medicine, we are studying functional effects of a miRNA SNP mutation found from Hypertrophic cardiomyopathy (HCM) patients. This project may lead to development of novel diagnosis and therapeutics for cardiovascular diseases including HCM in the future.

2. siRNA

Drosophila Dicer-2 associates with the dsRNA-binding partner proteins Loqs-PD and R2D2 and produces 21 nt long siRNAs from long dsRNA (Figure 2). siRNA is loaded to Argonaute2 and silences highly complementary target RNAs by cleaving them—a process typically called RNAi. One of the biological functions of the siRNA pathway is to fight against exogenously derived viral infection and against genome encoded transposon invasion. In addition, Dicer-2 produces endogenous siRNAs (endo-siRNAs) derived from genome encoded long hairpin RNA or overlapping mRNAs. The biological functions of these classes of endo-siRNAs are not well understood. We are interested in how viral and endogenously derived RNAs are recognized and cleaved into siRNAs by Dicer-2 and how the produced siRNAs function in biology. We also try to identify and characterize novel factors involved in or regulating the siRNA pathways. We are also interested in understanding how the two Dicer enzymes achieve their respective substrate specificities (pre-miRNA for Dicer-1 and long dsRNA for Dicer-2). Recently, we found that physiological concentration of inorganic phosphate, a small molecule found in all the cells, restricts the substrate specificity of Dicer-2 to long dsRNA by inhibiting Dicer-2 from cleaving pre-miRNA, without affecting cleavage of long dsRNA (Figure 4). We propose that inorganic phosphate occupies the phosphate-binding pocket in Dicer-2 and thereby block access of pre-miRNA. Currently we are investigation the function of the phosphate-binding pocket.

3. piRNA

piRNAs (26-31 nt) are mostly produced in gonads (ovaries and testes). Unlike miRNAs and siRNAs, Dicer enzymes are not involved in the piRNA production. piRNAs are produced in the primary processing pathway and the ping-pong pathway, which are not yet fully understood (Figure 5). piRNAs are loaded onto PIWI proteins and function in epigenetic and post-transcriptional gene silencing of transposons and other genetic elements in order to maintain genome integrity of germline cells. Interestingly, piRNAs are recently implicated also in sex determination, neuronal functions in brain, and tumorigenesis in cancer cells. We are interested in the mechanisms for biogenesis and function of piRNAs. We are trying to identify new factors involved in the piRNA pathway, using a fly reporter system.

4. RNA helicase

RNA helicases are involved in almost all the aspects in the RNA biology: RNA transcription, transport, translation, silencing, localization, structural rearrangement, decay, and so on. The Dicer enzymes also have a N-terminal 'helicase' domain. We are studying molecular and physiological roles of DEAD-box RNA helicases. Particularly, we are currently focusing on Drosophila belle, a DEAD-box RNA helicase that essential for fly viability and fertility and is conserved from yeast to human (Figure 6). We are making various mutant Belle and analyzing them genetically and biochemically.

Summary

Our lab uses multi-disciplinary approaches to understand the biogenesis and function of small silencing RNAs from the atomic to the organismal level. Small silencing RNAs play crucial roles in various aspects in biology. In fact, mutations in the small RNA genes or in the genes involved in the pathways cause many diseases in human including cancers. Our research projects will answer fundamental biological questions and also potentially lead to therapeutic application to human disease.

Positions available

Postdoc and student positions are available. Please contact the PI if interested.

Erin GoleyAssistant Professor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

The bacterial cell, once viewed as lacking internal organization, is in fact exquisitely structured at the subcellular level, and this spatial organization is critical for cellular survival and reproduction. As in eukaryotes, prokaryotic cell shape and internal structure are defined and dynamically remodeled by cytoskeletal elements. These proteins play critical roles in essential processes like cytokinesis and chromosome segregation, making them attractive targets for the development of novel antibiotics. Nevertheless, our understanding of the physiological functions of bacterial cytoskeletal proteins and the mechanisms by which they carry out their roles is still in its infancy. Our lab focuses on investigating cytoskeletal processes in bacteria, with current effort concentrated on understanding cytokinesis in the model bacterium, Caulobacter crescentus. The mechanisms underlying cell cycle progression and development are well-characterized in Caulobacter, making it an ideal system to address cytoskeletal function in the context of a well-defined cell cycle regulatory paradigm.

Cytoskeletal function during division: To tackle the question of how bacterial cells divide, we focus primarily on the function and regulation of the highly conserved tubulin-like protein, FtsZ. FtsZ is thought to act as a scaffold for assembly of the cytokinetic machinery, to generate constrictive forces that drive division, and, ultimately, to direct remodeling of the cell wall. However, the molecular details of FtsZ function are largely unknown. To gain insight into the mechanisms and regulation of bacterial growth and division, we are asking questions such as:

• How do the superstructure and dynamics of FtsZ polymers relate to its function?

• How is FtsZ attached to the membrane, and how does it generate force?

• How does FtsZ direct remodeling of the cell wall?

• How is the activity of FtsZ is regulated over the cell cycle?

We take a multi-faceted approach to address these questions, combining bacterial genetics, microscopy, biochemistry, and in vitro reconstitution to obtain a comprehensive view of the mechanisms of FtsZ action. These studies will inform models for how proteins at the division site direct cell growth and division and how these processes are integrated with other cell cycle events in time and space. In light of the high degree of conservation of cell division proteins among bacteria, our results will be relevant to the vast majority of bacterial species, including important human and animal pathogens.

Seth MargolisAssistant Professor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

Synapses are specialized cell-cell junctions which connect individual neurons together and are the sites of transmission of information between neurons.While the molecular mechanisms which promote synapse formation have been a subject of intense investigation, little is known about the molecular mechanisms that limit synapse formation so that synapses form at the right time and place and in the correct numbers.We hypothesize that this step in the refinement of synaptic formation is crucial for the fine-tuning of neuronal connectivity and that signaling networks which limit synapses during development are either defective or inappropriately activated in cognitive disorders.Accordingly, our laboratory studies the signaling pathways that regulate synapse formation during normal brain development to begin to understand how, when these pathways go awry, human cognitive disorders develop.

Currently projects include studies of:

1) Ephexin5:Ephexin5 is a guanine nucleotide-exchange factor (GEF) that activates the small G-protein RhoA, a regulator of the actin cytoskeleton.Genetic loss- and gain-of-function studies indicate that Ephexin5 acts to restrict spine growth and synapse development in the developing brain.Upon induction of EphrinB/EphB ligand-receptor signaling, Ephexin5 is rapidly phosphorylated in an EphB-dependent manner and targeted for proteasome-dependent degradation.These findings suggest that Ephexin5 functions as a barrier to excitatory synapse development until its degradation is triggered by EphrinB binding to EphBs.Interestingly, the degradation of Ephexin5 is mediated by Ube3A, a ubiquitin ligase whose expression level is altered in the human cognitive disorder Angelman Syndrome (AS) and in some forms of autism. This suggests that aberrant EphB/Ephexin5 signaling during synaptic development may contribute to the abnormal cognitive function observed in AS and autism.

Using Ephexin5 our laboratory will pursue an understanding of the molecular pathways that regulate restriction of excitatory synapse formation and their relevance to the pathophysiology of Angelman Syndrome by addressing the following questions:

1) What are the molecular determinants critical for Ube3A-mediated control of Ephexin5 degradation?

2) What molecular and cellular events underlie Ephexin5-mediated excitatory synapse restriction important for basic wiring of the nervous system?

3) What additional substrates of Ube3A are important for synapse formation?

2) New regulators of synapse formation:The goal of this study will be to identify additional components of the genetic program that restrict synapse numbers using previously developed immunocytochemistry-based assay for neuronal synaptic connections in vitro.Specific targets will be corroborated using electrophysiological and in vivo morphological measurements.We are particularly interested in genes whose products function to restrict synapse formation early in development and are suggested to be defective or inappropriately activated in cognitive disorders.

Mollie K. MeffertAssociate Professor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

Our laboratory is particularly interested in how changes in synaptic activity are converted into long-term alterations in the function and connectivity of neurons through the modulation of gene expression. Fundamental questions in gene expression of interest to the lab include:

Why are changes in gene expression required for enduring alterations in synaptic strength, such as during learning, development, or disease?

Study of the NF-κB transcription factor provides a good vantage point from which to explore transcriptional regulation in neurons. NF-κB has emerged as a key player in many CNS diseases, including neurodegenerative disorders and cancer. In the healthy CNS, studies from multiple laboratories including our own have demonstrated an evolutionarily conserved requirement for NF-κB in learning and memory. NF-κB is present at synapses and can undergo activation and nuclear translocation from distal processes upon synaptic stimulation. A current focus of our lab is to understand the signaling by the synaptic pool of NF-κB and how NF-κB regulates neuronal functions in both plasticity and disease.

Gene expression in the nervous system can be rapidly altered by control at the level of translation. Changes in translation, like transcription, are also critical for long-term information storage. A second major focus of our laboratory investigates how target specificity is generated in response to neuronal stimuli that regulate protein synthesis. We have discovered that the translating pool of RNA may be controlled through both positive and negative regulation of the biogenesis of mature microRNA from precursor microRNA. Ongoing investigations in our laboratory are aimed at further exploration of the importance of micoRNA biogenesis in determining rapid and specific changes in the neuronal and synaptic proteome and the in vivo roles of these pathways in healthy and dysregulated brain function.

Tamara O'ConnorAssistant Professor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

The outcome of most parasitic relationships is decided by an elaborate series of events involving hundreds of proteins. Understanding this interaction requires the analysis of the molecular mechanisms operating in both organisms and the causal relationships acting at the interface between them. The O’Connor lab studies the molecular basis of infectious disease using Legionella pneumophila pathogenesis as a model system.

L. pneumophila is a bacterial pathogen. In its natural environment of fresh water and soil, L. pneumophila is a parasite of a diverse array of amoebae and ciliated protozoa. When contaminated water aerosols are inhaled by humans, L. pneumophila replicates in alveolar macrophages causing an often fatal form of pneumonia called Legionnaires’ disease. We examine how L. pneumophila is able to manipulate host cell processes to establish growth within its host and the impact of its interaction with protozoa on the evolution of these virulence mechanisms.

The network of molecular interactions acting at the host-pathogen interface

Many bacterial pathogens use sophisticated secretion systems to translocate bacterial proteins into the cytoplasm of their host cell. These proteins modulate a vast array of host cellular processes to promote bacterial survival and replication. Defining the molecular mechanisms by which these proteins contribute to virulence is essential in understanding how bacterial pathogens cause disease.

L. pneumophila harbors one of the largest repertoires of translocated substrates identified to date, deploying an arsenal of 270 proteins to modulate host cell possesses. Maintaining this large repertoire has resulted in a high degree of redundancy whereby different proteins can manipulate complementary host cell pathways providing the bacterium with multiple strategies to accomplish a single task. We use L. pneumophila pathogenesis to examine the numerous mechanisms by which an intracellular bacterial pathogen can establish infection, how it exploits host cell machinery to accomplish this and how individual proteins and their component pathways coordinately contribute to disease.

The role of environmental hosts in the evolution of human pathogens

Although the role of protozoa in the lifecycle of many bacterial pathogens is only beginning to be appreciated, it is emerging as an important aspect in the epidemiology of many water and soil-borne pathogens. Not only do protozoa function as natural reservoirs for many pathogenic microorganisms, their interaction both provides a rich environment for the evolution of novel virulence strategies and enhances invasiveness in mammalian hosts. The genetic and molecular characterization of this interaction is instrumental in understanding how bacterial pathogens persist in nature and the selective pressures that shape the evolution of virulence strategies that promote disease in humans.

L. pneumophila's ability to grow in a diverse array of protozoan hosts allows us to examine the molecular determinants of host-range and the evolution of microbial pathogenesis in nature. In fresh water and soil, L. pneumophila is destined to encounter a large number of amoebal species. This necessitates a virulence strategy that can compensate for host variation and thus, the accumulation of host-specific virulence factors. Using genetics and functional genomics, we compare and contrast the repertoires of virulence proteins required for growth in a broad assortment of hosts, how the network of molecular interactions differs between hosts and the mechanisms by which L. pneumophila copes with this variation. By examining how virulence strategies acquired for growth in amoebae can be used in mammalian hosts, we are defining how this interaction promotes the transition of bacterial pathogens from their environmental reservoirs to humans, bridging the gap between the ecology of microbial pathogens and disease.

Peter PedersenProfessor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

In addition to studies focused on elucidating the structure, mechanism, and regulation of the mammalian mitochondrial ATP synthase, a major disease focus of the Pedersen lab for many years has been cancer because of its well known alterations in energy metabolism. More recently, we have entered also into a study of heart dysfunction as the heart with every beat is totally dependent on energy metabolism, with the mitochondrial ATP synthase being intimately involved.

The laboratory uses chemistry, molecular biology, biophysics, immunology, tissue culture and animal models to better understand the energetics/energy metabolism of normal and pathological cells/tissues. A major focus is on the two 'power plants', the mitochondria and the glucose catabolic system (glycolysis), as well as on the interaction between these two systems. The following are active research projects.

1) The mechanism and regulation of ATP synthesis in mammalian mitochondria.

This involves the study of the molecular properties of the ATP synthase complex that consists of two nano-motors both of which are necessary to make ATP. In a collaborative study we have obtained the 3-D structure of one of the motors and are now working on the structure of the whole complex that consists of 17 subunit types and over 30 total subunits.

Recently, we discovered that the ATP synthase is in complex formation with the transport system (carrier) for phosphate and the transport system for adenine nucleotides (ADP and ATP). We have named this complex the ATP Synthasome and are now carrying out studies to obtain a 3-D structure of the whole complex. It is important to note that the ATP synthasome represents the terminal complex of oxidative phosphorylation in mitochondria and makes most of the ATP needed/day to supply our energy needs.

In addition to the above mentioned work, we have also recently discovered that the ATP synthasome contains another key protein originally thought to be within the outer membrane as well as at contact sites between inner and outer membranes. This protein is likely critical for channeling ATP to the cytoplasm.

Work on the ATP synthasome is being vigorously studied.

2) Cancer: Regulation and targeting genes and proteins responsible for the most common phenotype and developing a novel potent anticancer agent, 3-bromopyruvate (3BP).

The most common metabolic phenotype of malignant cells & tumors including those derived from liver, breast, lung, brain, etc. is their capacity to utilize glucose at high rates even in the presence of oxygen. The pivotal enzyme involved is hexokinase 2 (HK-2) that is markedly elevated and bound at or very near the outer mitochondrial membrane protein named "VDAC" (voltage dependent anion channel). At this location, hexokinase 2 not only helps couple ATP formation in mitochondria to the phosphorylation of glucose to "jump start"glucose catabolism, it also represses this organelle's contribution to cell death. Therefore, hexokinase 2, in addition to its critical metabolic role, also promotes cancer by helping immortalize cancer cells. We are studying both the hexokinase 2 gene and developing novel strategies to target both the gene and the protein. We use both tumor cells growing in tissue culture and animal models, i.e., animals with cancer.

While working in my laboratory at the beginning of this century Dr. Young Ko discovered that the small molecule 3-bromopyruvate (3BP) is a potent anticancer agent. Several years later while working as a new faculty member in collaboration with my laboratory she would lead a team that showed 3BP's capacity to completely cure (eradicate) cancers in 19 out of 19 treated animals, i.e.,100%.

Currently, in collaboration with Dr. Ko, we are now involved in the further development of 3BP while searching for other effective anticancer agents. A limited number of studies conducted in humans with 3BP have proved very promising.

3) Heart Dysfunction: Regulation of the mitochondrial ATP synthase in the normal and ischemic heart.

The heart can survive only short periods without oxygen. Conditions where oxygen is limiting can have grave consequences as the mitochondrial membrane potential will collapse and the mitochondrial ATP synthase will switch from synthesizing ATP to hydrolyzing ATP, thus depleting heart cells (cardiomyocytes) of the energy reserves they require for survival. Fortunately, the ATP synthase is well regulated in the heart so that the ATP hydrolytic event is minimized during short periods of ischemia (reduced oxygen). In fact, there are 3 known small peptide regulators of the ATP synthase, one which optimizes ATP synthesis and the other two that suppress ATP hydrolysis. In addition, the ATP synthase is subjected to regulatory signal transduction events that result either in its phosphorylation or dephosphorylation.

We are currently involved in a project designed to understand the relative importance of these and other regulatory events in protecting the heart during sudden ischemic insults. [The laboratory has published over 240 papers of which >150 describe original research while the others refer either to novel methods or represent reviews]

Joel L. PomerantzAssociate Professor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

Our laboratory studies the molecular machinery used by cells to interpret extracellular signals and transduce them to the nucleus to effect changes in gene expression. This process is of fundamental biological importance. The accurate response to extracellular signals results in a cell's decision to proliferate, differentiate, or die, and it is critical for normal development and physiology. The disregulation of this machinery underlies the unwarranted expansion or destruction of cell numbers that occurs in human diseases like cancer, autoimmunity, hyperinflammatory states, and neurodegenerative disease.Currently, we study signaling pathways that are important in innate immunity, adaptive immunity, and in cancer, paying particular attention to pathways that regulate the activity of the pleiotropic transcription factor NF-kB. We are interested in these broad questions:

What are the biochemical mechanisms of signal transduction?

How is the input-output specificity determined so that each particular ligand or extracellular cue induces the appropriate cellular response?

How does the molecular specificity at the atomic level underlie biological specificity at the organismal level?

How are signaling pathways disregulated in human disease and can we use this knowledge to develop new therapeutics?

Can we use our understanding of signaling mechanisms to design novel, artificial signaling circuits for research and therapeutic purposes, for example, to control cell fate?

Examples of current projects:The biochemistry of antigen receptor signaling in B and T lymphocytes.The activation of NF-kB by antigen receptor engagement is a critical requirement for the activation of lymphocytes in the adaptive immune response. Using a novel expression cloning strategy designed to isolate molecules that signal to NF-kB in lymphocytes, we cloned CARD11, a multiprotein adaptor molecule and member of the MAGUK family of signaling proteins. We demonstrated that CARD11 plays a pathway-specific, factor-specific role in the activation of NF-kB downstream of T cell receptor signaling (Pomerantz, Denny, and Baltimore, 2002). We are currently investigating the biochemical mechanisms by which CARD11 transduces signals from the T cell receptor to NF-kB.Expression cloning of signaling molecules that regulate NF-kB, NFAT, and other transcription factors.We have used our expression cloning strategy (Pomerantz, Denny, and Baltimore, 2002) to clone several novel signaling molecules that signal the activation of the NF-kB or NFAT transcription factors. We will study their biological roles and characterize their mechanisms of action. We are also investigating whether our protocol is adaptable for the isolation of signaling molecules that regulate other transcription factors that influence the decision to proliferate, differentiate, or die, and that are disregulated in human disease.Design of novel signal transduction pathways for cell engineering.We are interested in testing our understanding of signal transduction by applying mechanistic insights toward the design of novel artificial cellular circuits. Our goal is to develop heterologous circuitry that would provide new tools for controlling gene expression to be used in biological research and to engineer cell fate decisions in novel therapeutic approaches.

Natasha ZacharaAssociate Professor
of Biological Chemistry
Johns Hopkins University School of Medicine

Program Description

Hundreds, if not thousands, of key cellular proteins in the nucleus, mitochondria and cytoplasm of metazoans are modified by O-linked β-N-acetylglucosamine (O-GlcNAc). Deletion of the UDP-GlcNAc: polypeptide O-β-N-acetyl-glucosaminyltransferase (OGT), the enzyme that adds O-GlcNAc, is lethal in animals and single cells highlighting the importance of this simple post-translational modification. O-GlcNAc is thought to act as a modulator of protein function, in a manner analogous to protein phosphorylation; the addition of O-GlcNAc to the protein backbone is dynamic, responding to morphogens, the cell cycle, changes in glucose metabolism, and cellular injury. O-GlcNAc occurs at sites on the protein backbone that are similar to those modified by protein kinases; and is reciprocal with phosphorylation on some well studied proteins, including RNA Pol II, estrogen receptor-β, SV-40 large T-antigen, endothelial nitric oxide synthase, and the c-Myc proto-oncogene product. These data suggest that one mechanism by which O-GlcNAc modulates cellular function is by competing with phosphorylation. A clear role for O-GlcNAc in cellular regulation has not emerged, although modulation of O-GlcNAc levels are implicated in the etiology of Type II Diabetes, cancer, and neurodegenerative diseases.

In response to multiple forms of cellular stress, levels of the O-GlcNAc protein modification are elevated rapidly and dynamically on myriad nuclear, mitocohdrial and cytoplasmic proteins. Several studies demonstrate that elevation of O-GlcNAc prior to heat stress, oxidative stress, hypoxia, trauma hemorrhage, and ischemia reperfusion injury is protective, suggesting that increased O-GlcNAc in response to stress is a survival response of cells injury. However, the mechanisms by which O-GlcNAc regulates protein function leading to cell survival have not been defined. Our long-term goal is to determine how stress-induced changes in the O-GlcNAc protein modification lead to increased cell/tissue survival in response to injury, in order to develop novel strategies for the treatment of numerous diseases, including ischemia reperfusion injury. Current research in the lab focus's on: 1) Characterizing the molecular mechanisms by which O-GlcNAc regulates heat shock protein expression; 2) The development of novel cells lines and tools for studying the O-GlcNAc modification; 3) Identifying proteins that are O-GlcNAc modified in response to different forms of cellular injury; 4) Understanding the signal transduction pathways that regulate O-GlcNAc modification in response to cellular injury; 5) Determining how O-GlcNAc regulates other stress-induced signaling pathways such as protein-phosphorylation. Together these studies will define a molecular road map from which we, and others, can determine the mechanism(s) by which O-GlcNAc promotes cell survival in diverse models, highlighting new targets for the development of alternative strategies that enhance stress-tolerance and promotes survival relevant models such as ischemic reperfusion injury. In addition, these studies will form a foundation for determining how dysregulation of the “O-GlcNAc-mediated stress response” contributes to pathologies such as type II diabetes and aging.